Methods and active substances for protecting organs

ABSTRACT

The present invention comprises a method of protecting organs or tissue susceptible to reperfusion-induced dysfunction after ischemia. The method comprises parenterally administering to a patient a therapeutical composition containing natural alpha-1 acid glycoprotein, natural alpha-1 antitrypsin or a mixture thereof. Alternatively, organ or tissue transplants can be contacted with natural alpha-1 acid glycoprotein, natural alpha-1 antitrypsin or mixtures by perfusing or flushing them with a solution containing natural alpha-1 acid glycoprotein, natural alpha-1 antitrypsin or mixtures thereof in a concentration of 0.1 to 5 g/l.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Divisional of co-pending application Ser. No.09/956,606, filed on Sep. 18, 2001, the entire contents of which arehereby incorporated by reference and for which priority is claimed under35 U.S.C. § 120.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to protection of organs, tissues and organfunctions during reperfusion of ischemic organs. In particular, theinvention relates to prevention of functional impairment of organscaused by ischemia-reperfusion during organ transplantation, surgicaloperations in general, thrombolytic therapy, after major blood loss andother causes of shock and hypoperfusion of organs.

2. Description of Related Art

Prolonged ischemia followed by reperfusion induces apoptosis andinflammation leading to tissue damage and organ dysfunction, which iscalled ischemia-reperfusion (I/R) injury or reperfusion injury. I/Rinjury takes place in various clinical conditions characterized bytemporary decrease or complete stop of blood flow to one or severalorgans (ischemia) followed by restoration of blood flow (reperfusion)(for review, see Collard, C. D. and Gelman, S., Anesthesiology94:1133-1138, 2001)

Ischemia-reperfusion injury accompanying organ transplantations leads todysfunction of the transplanted organ. When removed from the donor,organ transplants are perfused with cold preservation solution andsubsequently either stored cold or perfused with cold preservationsolution. This leads to cold ischemia in the organ transplant. Aftertransplantation to the recipient and restoration of blood flow,different extent of I/R injury develops in the organ transplant. Inkidney transplantation, I/R injury and concomitant renal dysfunctionleads to prolonged dependence on hemodialysis, whereas in heart, liverand lung transplantations immediate proper functioning of the graft iseven more important and graft dysfunction may lead to death of thepatient. The continuously increasing demand of donor organs necessitatesthe transplantation of organs from marginal donors with impaired bloodflow, including so called non-heart beating donors, whose organs arealways subjected to prolonged ischemia. This further contributes to I/Rinjury after transplantation.

Several preservation solutions aiming at minimizing tissue damage in theorgan transplants during hypothermal storage have been described. BelzerUW solution disclosed in U.S. Pat. Nos. 4,798,824 and 4,879,283 hasproved useful for all organ transplants, both for in situ organperfusion and cooling in the donor and for cold storage after the organis harvested. While the Belzer UW solution and some other preservationsolutions, such as the Euro-Collins solution (Squifflet J. P. et al.,Transplant. Proc. 13:693-696, 1981), have been effective in extendingthe cold preservation time of organs intended for transplantation,tissue injury during cold storage and particularly during reperfusionstill occurs. Therefore, reduction in I/R injury and concomitantdysfunction of organ transplants is desirable. Other preservationsolutions for organ perfusion and storage are disclosed in U.S. Pat.Nos. 4,415,556, 5,145,771, 5,693,462, 6,045,990 and 6,110,504, but noneof them has addressed the protection of organ transplants against I/Rinjury.

Another clinical condition associated with I/R injury is impairment ofblood supply to a local anatomical area caused by occlusion of the bloodvessel by a blood clot (thrombosis). Thrombosis of coronary and brainarteries is a leading cause of death. With thrombolytic (fibrinolytic)therapy the blood clot can be dissolved and blood flow restored, therebypreventing necrosis of the tissues. However, fibrinolytic therapy may beassociated with I/R injury and concomitant organ dysfunction withpotentially serious clinical complications. Other revascularisationprocedures, such as percutaneous transluminal angioplasty and coronaryartery bypass surgery may also lead to I/R injury (Maxwell S. R. and LipG. Y., Int. J. Cardiol. 58:95-117, 1997).

A further clinical condition associated with temporarily decreased bloodflow to tissues and organs comprises surgical operations, such ascardiac surgery with or without cardiopulmonary bypass and angioplasticsurgery. For example, cardiac and aortic surgery may result in temporaryimpairment of blood flow to the kidneys, which results to I/R injury andrenal dysfunction.

Hypoperfusion of organs takes also place in various forms of shock, suchas caused by excessive bleeding. Upon restoration of adequate bloodflow, e.g. by restoring the circulating blood volume, I/R injury maytake place (Collard and Gelman, 2001).

Currently, there are no effective ways in clinical practise to preventI/R injury other than restricting the ischemia period to as short aspossible (Collard and Gelman 2001). As evident from the prior art,prevention of I/R injuries would be of utmost clinical importance in theprevention of dysfunction of critical organs in various clinicalconditions. Therefore, the present invention aims at providingtherapeutic means to prevent I/R injury and consequent organdysfunction.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method ofpreventing reperfusion-induced organ dysfunction in different clinicalconditions associated with ischemia.

It is a second object of the present invention to provide a method ofpreventing I/R injury associated with organ transplantations, surgicaloperations in general, thrombolytic therapy and revascularisationprocedures of any type of infarction, and treatment of bleeding shockand other forms of shock.

It is a third object of the invention to provide a novel preservationfluid which, when contacted with the organ or tissue which is to beimplanted into a recipient patient, is capable of preventing organ andtissue transplants.

It is a fourth object of the present invention to provide a novelpharmaceutical composition which, when administered to the recipient,prevents I/R injury of organ transplants.

It is a fifth object to provide a method and a novel pharmaceuticalcomposition for preventing I/R injury of a patient undergoing majorsurgery, such as cardiac and angioplastic surgery.

It is a sixth object to provide a method and a pharmaceuticalcomposition for preventing I/R injury of patients undergoingthrombolytic therapy or revascularisation procedures or treatment ofbleeding shock or other forms of shock.

The invention is based on the surprising finding that two plasmaproteins, viz. alpha-1 acid glycoprotein (AGP) and alpha-1 antitrypsin(AAT), effectively prevent reperfusion-induced organ dysfunction afterischemia. When AGP or AAT or mixtures thereof are administered to ananimal, in which the blood flow to an organ is temporarily closed toinduce ischemia and subsequently the blood flow is restored, the I/Rinjury which otherwise develops to the organ is effectively prevented.In particular, the AGP or AAT proteins used are plasma-based, i.e.isolated and purified from blood plasma of mammals, in particularhumans, or they comprise transgenic or recombinant alpha-1 acidglycoprotein or alpha-1 antitrypsin having an amino acid sequenceidentical or essentially identical with that of the corresponding humanglycoprotein. As will be discussed in more detail below, the AGP and AATused can be purified from human plasma or they can be produced byrecombinant DNA technology as transgenic proteins in animals or plantsor in cell culture. The glycosylation pattern of the AGP and AAT ispreferably similar to that of plasma-AGP or plasma-AAT, respectively.

According to the present invention the novel method of protecting organsor tissue susceptible to reperfusion-induced dysfunction after ischemia,comprises contacting the organs or tissue with an increasedconcentration of natural alpha-1 acid glycoprotein, natural alpha-1antitrypsin or a mixture thereof during the ischemia period orimmediately upon reperfusion, preferably no later than at the beginningof the reperfusion period to prevent apoptosis. The “contacting” cantake place both in vivo and in vitro. Thus, AGP and/or AAT can beparenterally administered to a patient, who has organs or tissuesusceptible to a condition of reperfusion-induced dysfunction afterischemia. On the other hand, it is also possible to carry out theinvention by perfusing organ or tissue transplants with a preservationfluid and store the organ or tissue transplants for a sufficient timeprior to the implantation of said organ or tissue in a patient requiringsuch implantation, whereby the preservation solution contains naturalalpha-1 acid glycoprotein, natural alpha-1 antitrypsin or mixturesthereof in an effective concentration. AGP, AAT or mixtures thereof canbe also added to a rinse solution used for flushing the organ or tissuetransplant before transplantation into the recipient. Other embodimentsof the invention will be discussed in more detail below.

The present invention provides several advantages. Being physiologicalplasma proteins, human AGP and AAT are not toxic or foreign proteins tohuman subjects. The administered amounts of AAT and AGP that areeffective in the prevention of I/R injuries result in plasmaconcentrations which are similar to those occurring in plasma duringinflammatory conditions. The plasma levels of AGP and AAT increaseconsiderably in inflammatory conditions, which is called acute phaseresponse. Whereas the rise in the level of endogenous AGP and AAT duringacute phase response takes place after a lack period and is too late toprevent the tissue injury that has already taken place, the presentinvention takes advantage of the possibility to increase rapidly AGPand/or AAT level in circulation and tissues by administrating purifiedexogenous AGP and/or AAT before tissue injury develops. Thus, it is apreferred embodiment of invention to increase the plasma concentrationof AGP or AAT or mixtures thereof to a level corresponding to thattypical for the acute phase response for a patient susceptible to acondition of inflammation.

According to the present invention it is possible to protect organtransplants by adding AGP and/or AAT to an artificial preservationfluid, which is used for perfusion of the organ transplant before coldstorage or for continuous perfusion during cold storage.

Human AGP is readily available in large volumes as a side product ofpresent blood fractionation, and the present invention provides animportant way of utilizing this source of the protein which to date hashad limited commercial value.

Next, the present invention will be examined more closely with the aidof the following detailed description and with reference to a number ofworking examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the prevention of reperfusion-induced apoptotic cell deathin ischemic kidney by AGP and AAT administration. Apoptosis was assessedby measuring caspase-1 and caspase-3 activities in kidney homogenates 24hours after reperfusion. The mice were given the treatments indicated atthe time point of reperfusion (t=0) or two hours after it (t=2). Datapresent mean±SEM, *P<0.05 vs. PBS-treated control animals.

FIG. 2 shows the inhibition of reperfusion-induced inflammatory reactionin ischemic kidney by AGP and AAT administration. Inflammatory reactionwas assessed by myeloperoxidase (MPO) determination 24 hours afterreperfusion, which indicates neutrophilic influx to the renal tissue.The mice were given the treatments indicated at the time point ofreperfusion (t=0) or two hours after it (t=2).

FIG. 3 shows the prevention of reperfusion-induced renal dysfunctionafter ischemia by AGP and AAT administration. Renal function wasassessed by the blood urea nitrogen (BUN) (A) and serum creatininedeterminations (B) 24 hours after reperfusion of the ischemic kidney.The mice were given the treatments indicated at the time point ofreperfusion (t=0) or two hours after it (t=2).

FIG. 4 shows the prevention of reperfusion-induced impairment of renalfunction after ischemia by human plasma AGP. AGP and PBS (phosphatebuffered saline) were administered to the mice at the time point ofreperfusion of the ischemic kidney and renal function was assessed bythe blood urea nitrogen (BUN) determination.

DETAILED DESCRIPTION OF THE INVENTION

Generally, the present invention comprises a method of protecting organsor tissue susceptible to reperfusion-induced dysfunction after ischemia,wherein said organs or tissue are contacted with an increasedconcentration of natural AGP, natural AAT or a mixture thereof. For thepurpose of the present invention, the term “increased concentration”stands for concentrations of said glycoproteins which are higher thanthose which the organ or tissue would otherwise encounter without theimplementation of the present invention. Thus, the organs or tissue canbe contacted with increased concentrations of AGP, AAT or mixturesthereof by parenterally administering about 20 to 500 mg/kg/day of saidglycoproteins to a patient, who has organs or tissue susceptible to acondition of reperfusion-induced dysfunction after ischemia, so as toraise the concentration of the proteins in the plasma. Usually, theplasma concentration is increased by at least 50%, preferably by atleast 100%, compared to the plasma concentration prevailing without theactive intervention during the implementation of the method of therapy.Typically, the concentration of AGP, AAT or a mixture thereof in theplasma of a patient is 2- to 6-times higher than before theintervention. For instance, the plasma concentrations of said proteinscan be raised to a level essentially corresponding to that occurring inplasma during inflammatory conditions. Depending on the patient and hisor her condition before the administration of AGP or AAT, thatconcentration can vary in the range of about 0.2 to 5 g/l.

The present invention shows that AGP and AAT effectively prevent I/Rinjury when administered parenterally. As evident from Examples 1 to 4,AGP and AAT effectively prevent reperfusion-induced apoptosis,inflammation and functional impairment in an ischemic organ. It is knownfrom prior studies (Daemen M. A. et al., J. Clin. Invest. 104:541-9,1999) that acute primary apoptosis during early reperfusion is crucialto the initiation of reperfusion-induced inflammation and development ofI/R injury. According to the present invention, AGP ant AAT administeredat time of reperfusion effectively prevent acute primary apoptosis, asindicated by assessment of apoptosis markers 2 hours after reperfusion(Example 1).

AGP and AAT administered at the time of reperfusion also effectivelyprevent inflammation and apoptosis 24 hours after reperfusion (Examples1 and 2). When AGP and AAT were administered 2 hours after theinitiation of reperfusion, they still prevented inflammation and lateapoptosis but clearly to a lesser extent. This suggests that AGP and AATmay have direct anti-inflammatory effects and can also preventTNF-α-dependent late apoptosis. However, according to the presentinvention these effects are less important in the prevention of I/Rinjury. As evident from Example 3, AGP and AAT administered at time ofreperfusion effectively protected against reperfusion-induced organdysfunction whereas their administration 2 hours after the initiation ofreperfusion failed to protect against organ dysfunction. This emphasizesthe importance of early administration of AGP and AAT and prevention ofacute primary apoptosis during reperfusion.

As far as the present invention is concerned, AGP and/or AAT should beadministered before, at or not later than 60 minutes after theinitiation of reperfusion. In particular it is preferred to administerAGP and/or AAT no later than 30 minutes after the initiation ofreperfusion.

AGP has previously been shown to protect against TNF-α-induced liverapoptosis in galactosamine-pretreated and actinomycin D-pretreated mice,whereas AAT conferred protection only in the galactosamine model (VanMolle W. et al., J. Immunol. 159:35555-35564, 1997). By contrast,according to the present invention, AGP and AAT effectively protectagainst the early primary apoptosis during reperfusion, which iscritical to the initiation of reperfusion-induced inflammation.Importantly, early primary apoptosis is not dependent on TNF-α. Further,according to the present invention prevention of the early apoptosis iscrucial for protection against reperfusion-induced organ dysfunctionafter ischemia. Additionally, the prior art is silent of the potentialtherapeutic use of AGP or AAT in the prevention of I/R injury.

In another prior study, a recombinantly engineered form of AGP enrichedwith sialyl Lewis^(x) (sLe^(x)) carbohydrate structures (sAGP) was shownto reduce local and remote injuries after intestinal ischemia in a ratmodel (Williams J. P. et al., Am. J. Physiol. 273:G1031-G1035, 1997).Although the article does not disclose the actual amount of sLe^(x) inthe sAGP, the concentration was “increased” which indicates that therecombinantly engineered sAGP protein did not correspond to plasma-AGP.sAGP reduced remote lung injury by 62% and complement-dependentintestinal injury by 28%. According to the reference, this protectionwas attributed to sLe^(x)-mediated inhibition of neutrophil andcomplement-mediated injuries. As discussed above, in the presentinvention, the AGP corresponds to natural plasma-AGP and thereforecontains very small amounts of sLe^(x), if any. Further, the presentinvention has shown that the protective effect of AGP is not dependenton sLe^(x) determinants (Example 4). Thus, it is possible to obtainvaluable and surprising results even with AGP which is essentially freeof sLe^(x).

In another important embodiment of the invention, to protect organtransplants, AGP and AAT can be added to the preservation fluid used forin situ organ perfusion and cooling in the donor and for cold storage orperfusion after the organ is harvested. The organ or tissue transplantscan be perfused or flushed with a solution containing AGP, AAT ormixtures thereof in a concentration of 0.1 to 5 g/l. Typically, theorgans or tissue are perfused with a solution containing, in addition toAGP, AAT or a mixture thereof, also at least one component selected fromthe group consisting of electrolytes and cell-protecting agents.

According to the present invention, human or similar natural AGPpreparation is used. The preparations according to the examples below,which proved to be effective in the prevention of I/R injury, have beenpurified from the plasma of healthy blood donors and contain only littlesLe^(x) determinants (about 10% of the protein). Further, we have foundthat removal of AGP molecules with possible sLe^(x) determinants byaffinity chromatography does not influence the protective effect ofnormal human AGP. This indicates that human AGP does not need to bereconstructed to contain increased levels of sLe^(x) determinants inorder to be effective in the prevention of I/R injury. On the contrary,according to the present invention, the AGP glycoproteins used are“natural” proteins in the sense that they contain no sLe^(x)determinants or possibly sLe^(x) determinants up to a levelcorresponding to that present in plasma AGP (i.e. a maximum of about 10%of the protein).

Furthermore, according to the present invention the effect of AGP andAAT is rapid and apoptosis is inhibited before any neutrophilinfiltration is observed, and an effect on leukocytes less important inprotection against reperfusion-induced organ dysfunction after ischemia.

In summary, when used for treating patients, AGP and AAT are, accordingto the present invention, administered by parenteral route, preferablyintravenously. It is preferred to administer them rapidly afterinitiation of reperfusion, most preferably at the time of reperfusion ofischemic organs. They can be given intravenously to the recipient beforeor after transplantation, preferably no later that during thetransplantation.

Effective concentrations of AGP and AAT in plasma are identical or belowthe levels occurring in human subjects during acute phase response. Thiscorresponds to plasma levels up to 5 g/l. Intravascular doses of AGP andAAT that are effective in the treatment of humans are in the range of20-500 mg/kg/day.

Turning now to the use of AGP and AAT in preservation or rinse solutionsit can be reiterated that by adding AGP and/or AAT to the preservationsolution used for organ perfusion and cooling in the donor and for coldstorage or perfusion after the organ is harvested, I/R injury in theorgan transplant can be prevented and functional recovery aftertransplantation promoted. AGP and AAT may be added to different types ofpreservation solutions, which typically contain electrolytes (such asNa⁺, K⁺, Mg⁺⁺, Cl⁻, SO₄ ²⁻, HPO₄ ²⁺, Ca²⁺ and HCO₃ ⁻) and may containvarious other agents protecting the cells during cold storage. Forexample, AGP and/or AAT can be added to the UW Belzer solution(ViaSpan®, DuPont Pharmaceuticals Company), which contains 50 g/lhydroxyethyl starch, 35.83 g/l lactobionic acid, 3.4 g/l potassiumphosphate monobasic, 1.23 g/l magnesium sulfate heptahydrate, 17.83 g/lraffinose pentahydrate, 1.34 g/l adenosine, 0.136 g/l allopurinol, 0.922g/l glutathionine, 5.61 g/l potassium hydroxide and sodium hydroxide foradjustment of pH to pH 7.4. Another example of a suitable preservationsolution is the Euro-Collins solution, which contains 2.05 g/lmono-potassium phosphate, 7.4 g/l dipotassium phosphate, 1.12 g/lpotassium chloride, 0.84 g/l sodium bicarbonate and 35 g/l glucose.These intracellular type preservation solutions are rinsed away from thedonor organ before completion of transplantation into the recipient byusing a physiological infusion solution, such as Ringer's solution, andAGP and/or AAT can be also added to a rinse solution. Further, AGPand/or AAT can be added to extracellular type preservation solutionswhich need to be flushed away, such as Perfadex (Vitrolife, Sweden),which contains 50 g/l dextran, 8 g/l sodium chloride, 400 mg/l potassiumchloride, 98 mg/l magnesium sulfate, 46 mg/l disodium phosphate, 63 mg/lpotassium phosphate and 910 mg/l glucose.

The novel preservation and rinsing solutions according to the presentinvention may have a composition essentially corresponding to any of thethree commercial solutions described above. However, the actualconcentrations of the conventional components may vary somewhat,typically within a range of about ±50%, preferably about ±30%, of themean values given above. Thus, to give an example, in a novelpreservation solution having a basic composition similar to the Belzersolution mentioned above, the concentration of hydroxyethyl starch mayvary in the range of about 25 to 75 g/l.

According to a preferred embodiment, to ensure maximum activity, AGP andAAT are added to a ready-made preservation or rinse solution just beforeuse. Alternatively, a suitable preservation solution containing AGPand/or AAT may be prepared beforehand.

By administrating AGP and/or AAT to a recipient of an organ transplantat time of transplantation, development of I/R injury in the organtransplant can be prevented. As a result of this, the function of theorgan transplant is more rapidly recovered, which is a prerequisite forthe success of the organ transplantation. In kidney transplantions, theprevention of renal dysfunction after transplantation decreasesdependence of the patient on hemodialysis. In liver, heart and lungtransplantations, the early proper function of the organ transplant iscritical and prevention of graft dysfunction should decrease mortalityof the patients. By adding AGP and/or AAT to the artificial preservationsolution used for organ perfusion and cooling and for cold storage, I/Rinjury in the organ transplant can be also prevented and functionalrecovery after transplantation promoted.

By administrating AGP and/or AAT to patients undergoing cardiac orangioplastic surgery, development of I/R injury during operation can beprevented. This decreases the need of postoperative critical care.Correspondingly, by administering AGP and /or AAT to patients undergoingthrombolytic therapy, development of I/R injury during reperfusion ofthe occluded vessel can be prevented and organ dysfunction can beavoided. In thrombolytic therapy of myocardial infarction this mayprevent cardiac arrythmias and cardiac insufficiency. In thrombolytictherapy of brain infarction, this may decrease neurological symptoms andpalsies. By administrating AGP and/or AAT to patients suffering frombleeding shock or other forms of shock, development of I/R injury duringrestoration of adequate circulation can be prevented and functionalrecovery of critical organs promoted.

According to an embodiment of the present invention, AGP and AAT andmixtures thereof are used in methods for preparing pharmaceuticalcompositions intended for use in any of the therapeutic methods oftreatment described above.

According to the present invention, the AGP and AAT used compriseglycoproteins having amino acid sequences identical or essentiallyidentical with those of human AGP and AAT, respectively. “Essentiallyidentical” refers here to polymorphic sequence variants of AGP and AAToccurring in healthy human subjects. AGP and AAT can be purified fromhuman plasma or they can be produced by recombinant DNA technology astransgenic proteins in animals or plants (cf. for example U.S. Pat. No.6,194,553, the contents of which is herewith incorporated by reference)or in cell cultures using bacterial, animal, plant or yeast cells (HuangJ. et al., Biotechnol. Prog. 17:126-133, 2001). According to the presentinvention, the therapeutic efficacy is not dependent on specificcarbohydrate structures of AGP or AAT, such the sLex determinant.Recombinant AGP and AAT do not need to have an identical glycosylationpattern with the corresponding protein purified from human plasma.

Preferably, the AGP and AAT used should carry a mammalian-typeglycosylation pattern.

Purification of AGP can be accomplished from Fraction V supernatant,which is a by-product when albumin is purified from human plasma by theCohn fractionation method. Being an exceptionally acid protein with a pIof about 2.7, AGP can be effectively purified from other contaminatingproteins by ion exchange chromatography. AGP can be bound to an anionexchange resin under conditions in which impurities are washed away, andpure AGP can be eluted from the resin. The functional group of the anionexchange resin can be diethylaminoethyl (DEAE),diethyl-(2-hydroxyprolyl)-aminoethyl (QAE) or quaternary ammonium (Q).Additionally, AGP can be contacted with a cation exchange resin underconditions in which impurities are bound to the resin and AGP isrecovered in the effluent. The functional group of the cation exchangeresin can be carboxymethyl (CM) or sulphonyl, such as sulphopropyl (SP).Other chromatographic methods can also be applied, such as hydrophobicinteraction chromatography, chelate affinity chromatography andadsorption chromatography.

A therapeutic preparation of AGP and AAT must be safe with respect topotential blood-borne viruses, which can be accomplished by having atleast one virus inactivation or removal step in the manufacturingprocess, which is effective against both enveloped and non-envelopedviruses, and by limiting the potential virus load in the starting plasmaby sensitive virus screening tests. The therapeutic preparations shouldhave a low endotoxin level (<0.1 IU/mg protein by the LAL test). Thepurity of plasma-derived AGP and AAT in the therapeutic preparationshould be at least 80% as studied by cellulose acetate or agarose gelelectrophoresis before possible addition of albumin or other stabilizerproteins. The purity of transgenic or recombinant AGP and AAT should beat least 99%.

AGP can be subjected to virus inactivation by several methods, such assolvent detergent (SD) method and pasteurization for 10 h at 60° C. SDtreatments are disclosed in the art, in particular in U.S. Pat. Nos.4,540,573, 4,764,369 and 4,820,805, the contents of which are herewithincorporated by reference. The organic solvent is preferably selectedfrom dialkylphosphates and trialkylphosphates having alkyl groups whichcontain 1 to 10 carbon atoms. Examples of trialkylphosphates aretri-(n-butyl)-phosphate, tri-(t-butyl)phosphate, tri-(n-hexyl)phosphateand tri-(2-ethylhexyl)-phosphate. The concentration of the solvent is inthe range of 0.01 g/l to 100 g/l, preferably about 0.1 g/l to about 10g/l, a typical concentration of tri-(n-butyl)-phosphate being about0.3%. The solvent can be used together with a non-toxic detergent, whichis capable of enhancing the effectiveness of the solvent. The SDchemicals can be removed from AGP for example by binding AGP to an anionexchange resin, removing SD substances by washing the resin and elutingthe AGP from the resin.

Another method for the elimination of possible blood borne viruses fromAGP is virus filtration. As AGP has a relatively small molecular weight(about 41 kD), it can be readily filtered with virus removal filterswith very small pore size, such as 15 nm, which effectively remove evensmallest non-enveloped viruses. Successful filtration with 15 nm virusremoval filters has been before described for plasma proteins, suchfactor IX and factor XI with molecular sizes of 55 kD and 143 kD,respectively (Burnouf-Radosevich M. et al., Vox Sang. 67, 132-138,1994).

In one purification method of AGP (Hao Y.-L. and Wickerhauser M.,Biophys. Biochim. Acta 322:99-108, 1973), Fraction V supernatant (pH4.7) is stirred with DEAE-Sephadex A-50 gel equilibrated with 25 mMsodium acetate buffer pH 4.1 for 90 min at 0 to 10° C. After washing thegel with the cold acetate buffer the adsorbed fraction is eluted withthe acetate buffer containing 1 M NaCl. The eluate is concentrated anddiafiltered with the acetate buffer by ultrafiltration. The diafilteredconcentrate is then applied to a column of CM-gel equilibrated with theacetate buffer and AGP is recovered in the column effluent. The pure AGPpreparation is concentrated and diafiltered against water for injection(WFI) or 150 mM sodium chloride by ultrafiltration. The solution isneutralized with 0.5 M NaOH and filtered through a virus filter withpore size of 15 nm. The solution is sterile filtered, and filledaseptically into sterile containers. The product can be lyophilized.

In another purification method, disclosed in more detail in publishedpatent application WO97/32893, Fraction V supernatant is applied to acolumn of Q Sepharose Big Bead equilibrated with 0.13 M sodium acetatebuffer pH 4.1 at 10° C. The column is washed with about 5 bed volumes ofthe acetate buffer and AGP eluted with the acetate buffer containing 0.2M sodium chloride. The AGP preparation is neutralized and treated for 2hours at 20° C. with Aerosil (1 g Aerosil per 1 g AGP) to removepossible pyrogens. The AGP preparation is concentrated byultrafiltration and diafiltered against 10 mM sodium phosphate, 150 mMNaCl, pH 7.5. The solution is filtered through a virus filter with poresize of 15 nm, sterile filtered and filled aseptically into sterilecontainers. The product is pasteurized in the final containers for 10hours at 60° C.

By both described methods, virus-safe and pure AGP having a low pyrogencontent and being suitable for intravascular administration to humans isobtained.

In summary, according to a preferred embodiment of the presentinvention, AGP is purified from Cohn fraction V supernatant of humanplasma by a method comprising anion exchange chromatography,ultrafiltration, virus filtration and at least one virus inactivationstep consisting of solvent detergent treatment or pasteurization.

AAT may be purified from human plasma starting for example from Cohnfraction IV precipitate by different described methods (cf. for exampleU.S. Pat. No. 5,610,285, the contents of which is herewith incorporatedby reference).

As apparent to a person skilled in the art, therapeutic compositions ofAGP and AAT may contain various known excipients and stabilizers. Theymay also be combined with each other or given at the same time asseparate compositions. Further, they can be combined either in the samecomposition or as different compositions with other active substances inthe treatment of clinical conditions associated with I/R injury.

Typically, therapeutically useful pharmaceutical compositions—preferablyformulated for parenteral use—contain AGP or AAT or mixtures thereof inconcentrations of 1-200 g/l, preferably about 10 to 100 g/l.Pharmaceutical compositions of AGP and AAT may also comprisefreeze-dried powder, which can be reconstituted to a suitable volumebefore administration to a patient or addition to a preservationsolution of organs or tissues.

The following non-limiting examples illustrate the invention:

EXAMPLE 1 AGP And AAT Reduce Early And Delayed Reperfusion-InducedApoptosis After Ischemia

To induce renal I/R, mice weighing 20-25 g were subjected to 45 minutesof unilateral ischemia of the left kidney by clamping the renal vesselsas described before (Daemen M. A. et al., Circulation 102:1420-1426,2000). Contralateral nephrectomy was performed and the clamp removed. Atreperfusion, 12 mice were administered intraperitoneally 5 mg bovine orhuman AGP or 0.5 mg human AAT in 0.5 ml sterile PBS, which resulted inserum concentrations similar to those observed during acute phasereaction. 8 mice received AGP or AAT 2 hours after reperfusion. 10control mice received 0.5 ml PBS and 12 sham-operated mice weresubjected to same procedure without clamping the renal vessels andtreated with PBS. The animals were euthanized at indicated time pointsand blood samples were collected and the left kidney harvested. The samemodel of I/R injury was used in all examples.

Development of reperfusion-induced apoptotic cell death in the kidneywas assessed by determination of intranucleosomal DNA cleavage,caspase-1 and caspase-3 activities and TUNEL microscopy as describedbefore (Daemen M. A. et al., Circulation 102:1420-1426, 2000). Nointernucleosomal DNA cleavage was detected at 2 hours of reperfusion inkidneys obtained from mice treated with either AGP or AAT, whereas clearDNA cleavage was observed in PBS-treated mice. These early effects ofAGP and AAT indicated direct inhibition of apoptosis as early primaryapoptosis precedes the first signs of inflammation during I/R injury. Asindicated by the absence of apparent internucleosomal DNA cleavage,decreased numbers of TUNEL-positive nuclei, and attenuated caspase-1 andcaspase-3 activities (FIG. 1), Apoptosis was also effectively reducedafter 24 hours in mice treated with either AGP or AAT compared withPBS-treated control mice.

The therapeutic effect was less when AGP and AAT were administered 2hours after reperfusion. Compared with PBS treatment, AAT administeredat 2 hours of reperfusion decreased caspase-1- and caspase-3-likeactivities after 24 hours of reperfusion (FIG. 1), but did not reduceinternucleosomal DNA cleavage. AGP reduced caspase-1 and caspase-3-likeactivities (FIG. 1) and prevented internucleosomal DNA cleavage after 24hours.

Dose response of the therapeutic effect of AGP was studied bydetermining internucleosomal DNA cleavage 2 hours after reperfusion andadministration of different doses of AGP. A single dose of 1.7 mg AGP atreperfusion sufficed to effectively reduce internucleosomal DNAcleavage. The therapeutic effect gradually declined when doses of 0.5 or0.17 mg AGP were used.

EXAMPLE 2 AGP And AAT Reduce Reperfusion-Induced Inflammation AfterIschemia

The effects of AGP and AAT on reperfusion-induced inflammation werestudied by assessing renal TNF-α expression and neutrophil influx. AGPand AAT administered at time of reperfusion effectively limited TNF-αexpression and neutrophil influx, studied at 24 hours after initiationof reperfusion (FIG. 2). AGP and AAT given after 2 hours of reperfusionattenuated the inflammation at 24 hours to a lesser extent than didtreatment given at time of reperfusion (FIG. 2). However, treatments atboth time points decreased inflammation compared with PBS treatment whenassessed 24 hours after reperfusion (FIG. 2). The PBS-treated controlmice showed significant renal inflammation as reflected by TNF-αexpression in the outer stripe of the outer medulla, along the damagedtubular epithelium, and in infiltrating leukocytes. Also, significantrenal neutrophil accumulation was reflected by an enhancedmyeloperoxidase (MPO) content (FIG. 2) and accumulation ofanti-neutrophil antibody-positive cells.

EXAMPLE 3 AGP And AAT Prevent Reperfusion-Induced Renal DysfunctionAfter Ischemia

Ischemia-reperfusion of the kidneys resulted in renal dysfunction asreflected by increased blood urea nitrogen (BUN) content and serumcreatinine levels 24 hours after reperfusion (FIGS. 3 A and B). Comparedwith PBS, both AGP and AAT administered at time of reperfusioneffectively lowered BUN content and serum creatinine levels. However,when administered 2 hours after reperfusion, AGP and AAT failed tosignificantly decrease BUN or serum creatinine compared with PBS (FIGS.3 A and B). These findings illustrate the importance of the preventionof early apoptotis after reperfusion and the necessity to administer AGPand AAT at the time point of reperfusion in order to achieve optimaltherapeutic effect in the prevention of I/R injury. Bovine AGP (FIG. 3),human plasma AGP (FIG. 3) and human AGP lacking the sLe^(x) carbohydratestructure had similar efficacy in preventing reperfusion-induced renaldysfunction after ischemia.

EXAMPLE 4 Preparation of AGP From Human Plasma

980 kg of Fraction V supernatant was obtained from 1500 kg of humanplasma during purification of albumin by Cohn fractionation. Thesupernatant was filtered through a Cuno Zeta Plus depth filter andstirred for 3 h at 4° C. with 80 l of pre-swelled DEAE-Sephadex A-50 gelequilibrated with 25 mM sodium acetate buffer, pH 4.1. The gel wasallowed to sediment, the supernatant siphoned away and the gel waswashed 5 times with 40 l of the acetate buffer. AGP was eluted from thegel with 160 l of the acetate buffer containing 1 M NaCl. The eluate wasconcentrated to 3000 ml using 30 kD cut-off ultrafiltration membranesand diafiltered against 25 mM sodium acetate buffer, pH 4.1. The AGPsolution was applied to a column of CM-Trisacryl M (bed volume 1.3 l)equilibrated and eluted with the 25 mM sodium acetate buffer, pH 4.1.AGP was recovered in the column effluent, concentrated to a proteinconcentration of 70 g/l by ultrafiltration and diafiltered against WFI.The pH was neutralized and the solution was filtered through a Planova15N virus filter. The solution was sterile filtered, filled asepticallyinto sterile containers and lyophilized. Before administration, theproduct was reconstituted with sterile 150 mM NaCl to a proteinconcentration of 10 g/l.

The purity of AGP in the product was more than 95% according tocellulose acetate electrophoresis. 97.5% of the protein was monomericAGP, 2.5% corresponded to dimers, and no polymers or aggregates weredetected by size exclusion HPLC. The endotoxin level was 0.02 IU/mgaccording the LAL-test (Ph. Eur. 3rd Ed. S. 2.6.14.A, 2000) and theproduct was free of pyrogens when tested at a dose of 100 mg/kg inrabbits.

A fraction of human plasma AGP containing sLe^(x) determinants wasremoved by affinity chromatography using agarose-bound Aleuria aurantialectin (De Graaf T. W. et al., J. Exp. Med. 177:657-666, 1993) accordingto the manufacturer's instructions (Vector Laboratories). AGP lackingthe sLe^(x) determinant was recovered in the effluent and thelectin-bound fraction was eluted with 100 mM fucose. About 10% of humanplasma AGP bound to the column.

1. A preservation solution for perfusion and storage of organtransplants comprising at least one electrolyte and 0.1-5 g/l of acell-protecting agent, wherein said cell-protecting agent comprises atleast one member selected from the group consisting of human alpha-1acid glycoprotein and human alpha-1 antitrypsin.
 2. The solutionaccording to claim 1, wherein the alpha-1 acid glycoprotein or alpha-1antitrypsin comprises human plasma-derived alpha-1 acid glycoprotein oralpha-1 antitrypsin having a purity of at least 80% or transgenic orrecombinant alpha-1 acid glycoprotein or alpha-1 antitrypsin having anamino acid sequence essentially identical with that of the correspondinghuman alpha-1 acid glycoprotein or human alpha-1 antitrypsin and havinga purity of at least 99%.
 3. The solution according to claim 1, whereinthe natural alpha-1 acid glycoprotein or natural alpha-1 antitrypsincomprises a virus-safe therapeutic composition having an endotoxin levelof less than 0.1 IU/mg protein as measured by the limulus amebocytelysates (LAL) test.
 4. The solution according to claim 1, wherein thehuman alpha-1 acid glycoprotein has been purified from Cohn fraction Vsupernatant of human plasma by a method comprising anion exchangechromatography, ultrafiltration, virus filtration and at least one virusinactivation step consisting of solvent detergent treatment orpasteurization.